Most living things including plants are easily affected by not only biological stresses such as pathogen, insect, virus, etc, but also in variety of environmental stresses such as high temperature, salt, drought, pollution, wound, cold injury, excessive light condition, ozone, sulfur dioxide, over-exposure of UV, osmotic shock, etc. When plants are affected by various environmental stresses like the above, oxygen therein, which is essential for life-support, changes into reactive oxygen free radicals including superoxide anion radical (O2−), hydrogen peroxide (H2O2), hydroxyl radical, etc, causing serious physiological disorders in vivo. Precisely, small amount of oxygen free radical in vivo is enough to transmit signal in cells and induce the expression of genes (antioxidant enzyme, heat shock protein, etc.) necessary for self-defense in a plant. However, the increase of reactive oxygen free radicals causes physiological disorders, by dose-dependent manner, even to cell death.
Recently, many researchers are deeply interested in signal transduction pathway mediated by oxygen free radicals in plants. That is because the regulation of that pathway enables the increase of the expressions of antioxidant enzyme and host-defense protein in cells and leads to the development of plants having strong resistance against any environmental stress (Kovtun, et al., Proc. Natl. Acad. Sci. USA, 97(6): 2940-2945, 2000). According to a recent report, MAP kinase cascade (MARK cascade) plays an important role in signal transduction pathway mediated by oxygen free radicals in plants (Kovtun, et al., Proc. Natl. Acad. Sci. USA, 97(6): 2940-2945, 2000).
Superoxide dismutase (SOD) is an enzyme that converts superoxide anion radical (O2−) into hydrogen peroxide (H2O2), and is divided into CuZnSOD, MnSOD and FeSOD according to metal cofactor included in the enzyme. These are located differently in cells, for example CuZnSOD is found in cytoplasm and chloroplasts, MnSOD is found in mitochondria and FeSOD exists in chloroplasts. SOD is an important environmental tolerant factor that eliminates oxygen free radicals generated in a living body by environmental stress, which can be useful for the production of medical supplies, food, cosmetics, etc. And thus, preparing a transgenic plant containing SOD gene showing a strong environmental stress tolerance leads to the development of a plant having a strong resistance to environmental stresses such as ozone, low temperature, herbicides, etc (Plant Physiology, 10: 1049-1054, 1995; U.S. Pat. No. 5,538,878).
Ascobate peroxidase (APX) is an enzyme that converts H2O2 into water by using ascobate as an electron donor, and is largely found in plants and insects. This enzyme is known to exist in cytoplasm, stroma in chloroplasts and thylakoid membrane in plants (Free Rad. Biol. Med. 23: 473-479, 1997).
In chloroplasts of a plant, oxygen content is relatively high and electron transport system is operating to utilize electronic energy produced from the decomposition of water with light energy, so this organ is very sensitive to various oxidative stresses. Thus, the increase of anti-oxidative capability of chloroplast might be a great help to maintain productivity of a plant under environmental stresses.
As of today, in order to develop a stress tolerant plant, CaMV 35S promoter, which is constitutively expressed regardless of conditions, has been mostly used for the combination with a gene with resistance for specific stress to construct an expression vector. So, the resultant plant has resistance against only a specific stress. In order to overcome this problem, it is required to prepare an expression vector including a promoter being able to be expressed under any stress circumstances and a stress tolerant gene, and a transgenic plant transfected with the vector.
In order to develop transgenic agricultural crops having tolerance against environmental disaster, the present inventors prepared a novel expression vector for plant transformation by attaching multiple stress tolerant genes SOD (superoxide dismutase) and APX (ascorbate peroxidase) to oxidative stress inducible peroxidase promoter SWPA2 originated from sweetpotato so as to express the genes in chloroplasts of a plant. And then, the inventors regenerated transformed plants prepared from potato, sweetpotato and tall fescue by tissue culture. At last, the present inventors completed this invention by confirming that the transgenic plants of the invention have increased multiple stress tolerance.